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The autoimmune type 1 diabetes (T1D) that arises spontaneously in NOD mice is considered to be a model of T1D in humans. It is characterized by the invasion of pancreatic islets by mononuclear cells (MNCs), which ultimately leads to destruction of insulin-producing β cells. Although T cell dependent, the molecular mechanisms triggering β cell death have not been fully elucidated. Here, we report that a glycosaminoglycan, heparan sulfate (HS), is expressed at extraordinarily high levels within mouse islets and is essential for β cell survival. In vitro, β cells rapidly lost their HS and died. β Cell death was prevented by HS replacement, a treatment that also rendered the β cells resistant to damage from ROS. In vivo, autoimmune destruction of islets in NOD mice was associated with production of catalytically active heparanase, an HS-degrading enzyme, by islet-infiltrating MNCs and loss of islet HS. Furthermore, in vivo treatment with the heparanase inhibitor PI-88 preserved intraislet HS and protected NOD mice from T1D. Our results identified HS as a critical molecular requirement for islet β cell survival and HS degradation as a mechanism for β cell destruction. Our findings suggest that preservation of islet HS could be a therapeutic strategy for preventing T1D. [ABSTRACT FROM AUTHOR]

Type 1 diabetes (T1D) is an autoimmune disease resulting from the destruction of insulin-producing beta cells in pancreatic islets. T lymphocytes are the claimed pathogenic effectors but abnormalities of other immune cell types, including neutrophils, also characterize T1D development. During human T1D natural history, neutrophils are reduced in the circulation, while accumulate in the pancreas where release of neutrophil extracellular traps (NETs), or NETosis, is manifest. Recent-onset T1D patients also demonstrate activated circulating neutrophils, associated with a unique neutrophil gene signature. Neutrophils can bind to platelets, leading to the formation of platelet-neutrophil aggregates (PNAs). PNAs increase in the circulation during the development of human T1D and provide a mechanism for neutrophil activation and mobilization/recruitment to the pancreas. In non-obese diabetic or NOD mice, T1D autoimmunity is accompanied by dynamic changes in neutrophil numbers, activation state, PNAs and/or NETosis/NET proteins in the circulation, pancreas and/or islets. Such properties differ between stages of T1D disease and underpin potentially indirect and direct impacts of the innate immune system in T1D pathogenesis. Supporting the potential for a pathogenic role in T1D, NETs and extracellular histones can directly damage isolated islets in vitro , a toxicity that can be prevented by small polyanions. In human T1D, NET-related damage can target the whole pancreas, including both the endocrine and exocrine components, and contribute to beta cell destruction, providing evidence for a neutrophil-associated T1D endotype. Future intervention in T1D could therefore benefit from combined strategies targeting T cells and accessory destructive elements of activated neutrophils., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Petrelli, Popp, Fukuda, Parish, Bosi and Simeonovic.)

Platelet-neutrophil aggregates (PNAs) facilitate neutrophil activation and migration and could underpin the recruitment of neutrophils to the pancreas during type 1 diabetes (T1D) pathogenesis. PNAs, measured by flow cytometry, were significantly elevated in the circulation of autoantibody-positive (Aab+) children and new-onset T1D children, as well as in pre-T1D (at 4 weeks and 10-12 weeks) and T1D-onset NOD mice, compared with relevant controls, and PNAs were characterized by activated P-selectin+ platelets. PNAs were similarly increased in pre-T1D and T1D-onset NOD isolated islets/insulitis, and immunofluorescence staining revealed increased islet-associated neutrophil extracellular trap (NET) products (myeloperoxidase [MPO] and citrullinated histones [CitH3]) in NOD pancreata. In vitro, cell-free histones and NETs induced islet cell damage, which was prevented by the small polyanionic drug methyl cellobiose sulfate (mCBS) that binds to histones and neutralizes their pathological effects. Elevated circulating PNAs could, therefore, act as an innate immune and pathogenic biomarker of T1D autoimmunity. Platelet hyperreactivity within PNAs appears to represent a previously unrecognized hematological abnormality that precedes T1D onset. In summary, PNAs could contribute to the pathogenesis of T1D and potentially function as a pre-T1D diagnostic.

Heparan sulfate proteoglycans (HSPGs) consist of a core protein with side chains of the glycosaminoglycan heparan sulfate (HS). We have previously identified (i) the HSPGs syndecan-1 (SDC1), and collagen type XVIII (COL18) inside mouse and human islet beta cells, and (ii) a critical role for HS in beta cell survival and protection from reactive oxygen species (ROS). The objective of this study was to investigate whether endoplasmic reticulum (ER) stress contributes to oxidative stress and type 2 diabetes (T2D) by depleting beta cell HSPGs/HS. A rapid loss of intra-islet/beta cell HSPGs, HS and heparanase (HPSE, an HS-degrading enzyme) accompanied upregulation of islet ER stress gene expression in both young T2D-prone db/db and Akita Ins2WT/C96Y mice. In MIN6 beta cells, HSPGs, HS and HPSE were reduced following treatment with pharmacological inducers of ER stress (thapsigargin or tunicamycin). Treatment of young db/db mice with Tauroursodeoxycholic acid (TUDCA), a chemical protein folding chaperone that relieves ER stress, improved glycemic control and increased intra-islet HSPG/HS. In vitro, HS replacement with heparin (a highly sulfated HS analogue) significantly increased the survival of wild-type and db/db beta cells and restored their resistance to hydrogen peroxide-induced death. We conclude that ER stress inhibits the synthesis/maturation of HSPG core proteins which are essential for HS assembly, thereby exacerbating oxidative stress and promoting beta cell failure. Diminished intracellular HSPGs/HS represent a previously unrecognized critical link bridging ER stress, oxidative stress and beta cell failure in T2D., Competing Interests: The authors declare that no competing interests exist. The donation by Progen Pharmaceuticals Limited of PI-88 compound for in vitro studies does not alter our adherence to PLOS ONE policies on sharing data and materials.”

Type 1 diabetes (T1D) results from autoimmune destruction of insulin-producing beta cells in pancreatic islets. The degradation of the glycosaminoglycan heparan sulfate (HS) by the endo-β-D-glycosidase heparanase plays a critical role in multiple stages of the disease process. Heparanase aids (i) migration of inflammatory leukocytes from the vasculature to the islets, (ii) intra-islet invasion by insulitis leukocytes, and (iii) selective destruction of beta cells. These disease stages are marked by the solubilization of HS in the subendothelial basement membrane (BM), HS breakdown in the peri-islet BM, and the degradation of HS inside beta cells, respectively. Significantly, healthy islet beta cells are enriched in highly sulfated HS which is essential for their viability, protection from damage by reactive oxygen species (ROS), beta cell function and differentiation. Consequently, mouse and human beta cells but not glucagon-producing alpha cells (which contain less-sulfated HS) are exquisitely vulnerable to heparanase-mediated damage. In vitro, the death of HS-depleted mouse and human beta cells can be prevented by HS replacement using highly sulfated HS mimetics or analogues. T1D progression in NOD mice and recent-onset T1D in humans correlate with increased expression of heparanase by circulating leukocytes of myeloid origin and heparanase-expressing insulitis leukocytes. Treatment of NOD mice with the heparanase inhibitor and HS replacer, PI-88, significantly reduced T1D incidence by 50%, impaired the development of insulitis and preserved beta cell HS. These outcomes identified heparanase as a novel destructive tool in T1D, distinct from the conventional cytotoxic and apoptosis-inducing mechanisms of autoreactive T cells. In contrast to exogenous catalytically active heparanase, endogenous heparanase may function in HS homeostasis, gene expression and insulin secretion in normal beta cells and immune gene expression in leukocytes. In established diabetes, the interplay between hyperglycemia, local inflammatory cells (e.g. macrophages) and heparanase contributes to secondary micro- and macro-vascular disease. We have identified dual activity heparanase inhibitors/HS replacers as a novel class of therapeutic for preventing T1D progression and potentially for mitigating secondary vascular disease that develops with long-term T1D.

Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing beta cells in pancreatic islets are progressively destroyed. Clinical trials of immunotherapies in recently diagnosed T1D patients have only transiently and partially impacted the disease course, suggesting that other approaches are required. Our previous studies have demonstrated that heparan sulfate (HS), a glycosaminoglycan conventionally expressed in extracellular matrix, is present at high levels inside normal mouse beta cells. Intracellular HS was shown to be critical for beta cell survival and protection from oxidative damage. T1D development in Non-Obese Diabetic (NOD) mice correlated with loss of islet HS and was prevented by inhibiting HS degradation by the endoglycosidase, heparanase. In this study we investigated the distribution of HS and heparan sulfate proteoglycan (HSPG) core proteins in normal human islets, a role for HS in human beta cell viability and the clinical relevance of intra-islet HS and HSPG levels, compared to insulin, in human T1D. In normal human islets, HS (identified by 10E4 mAb) co-localized with insulin but not glucagon and correlated with the HSPG core proteins for collagen type XVIII (Col18) and syndecan-1 (Sdc1). Insulin-positive islets of T1D pancreases showed significant loss of HS, Col18 and Sdc1 and heparanase was strongly expressed by islet-infiltrating leukocytes. Human beta cells cultured with HS mimetics showed significantly improved survival and protection against hydrogen peroxide-induced death, suggesting that loss of HS could contribute to beta cell death in T1D. We conclude that HS depletion in beta cells, possibly due to heparanase produced by insulitis leukocytes, may function as an important mechanism in the pathogenesis of human T1D. Our findings raise the possibility that intervention therapy with dual activity HS replacers/heparanase inhibitors could help to protect the residual beta cell mass in patients recently diagnosed with T1D.

Evidence for the in vivo transmission of porcine endogenous retrovirus (PERV) from porcine xenografts to various recipient animals has been inconsistent. To characterize the contribution of the host immune system to the potential for PERV transmission from pig islet tissue xenografts to host tissues, we examined two immunoincompetent animal models, thymectomizsed fetal lambs and NODscid mice. Pig proislets were grafted into fetal lambs or adult NODscid mice. Conventional, nested and real-time PCR/RT-PCR tests were used to search for PERV and pig cell-specific sequences (porcine mitochondrial cytochrome oxidase II (COII) or mitochondrial ribosomal 12S) in pig proislets, host liver and spleen at 5-84 days (lambs) or 96 days (mice) after transplantation. Xenografts were harvested at the same time points. The copy number of PERV sequences and host cell-specific nuclear (palmitoylcarnitine transferase) sequences was assessed by real-time PCR to estimate the proportion of PERV-infected host cells. Pig proislets were shown to be PERV+ve by PCR and immunohistochemistry (PERV B env protein p15E). PERV transmission (PERV A, B or C DNA in the absence of porcine COII or 12S sequences) was detected by nested PCR and real-time PCR in 4/12 fetal lamb liver samples 5-23 days after transplantation; the maximum copy number of PERV B env sequences was found at day 5 (700 copies/1 x 10(6) lamb cells). A total of 4/12 fetal lambs demonstrated both PERV and 12S porcine sequences in liver samples (days 5-84) by real-time PCR, suggesting that pig cells had migrated to those tissues and established microchimerism; nested PCR showed evidence for microchimerism (porcine COII sequences alone) in 2/12 lambs (day 5). The incidence of PERV transmission and frequency of microchimerism was similar in host spleen analysed by real-time PCR. Histological examination showed complete xenograft rejection by 23 days after transplantation to fetal lambs. In contrast, pig proislet xenografts survived long term (> or =day 96) in NODscid mice but no PERV transmission was found. Both nested and real-time PCR assays revealed that 2/3 mice had become microchimeric. Long-term expression of PERV A, B and C as well as porcine 12S or COII RNAs was found at the graft site (day 96) only, indicating that PERV transcription and possibly replication, continued in the donor pig islet tissue after transplantation. Overall, detection of PERV transmission and microchimerism was limited by the sensitivity of the PCR assay and the primers chosen. The absence of stable PERV transmission and microchimerism in fetal lambs and the rejection of pig proislet xenografts correlated in time with the establishment of host immunocompetence. We therefore suggest that the frequent failure to identify PERV transmission late after transplantation could be due to the immunological destruction of PERV-infected host cells. Recipient NODscid mice demonstrated long-term microchimerism and intragraft PERV expression, which was consistent with their stable immunoincompetence.

Background: Identification of the antigens that stimulate transplant rejection can help develop graft-specific antirejection strategies. The xenoantigens recognized during rejection of porcine cellular xenografts have not been clearly defined, but it has been assumed that major histocompatibility complex (MHC) xenoantigens are involved., Methods: The role of porcine endogenous retrovirus (PERV) as a source of xenoantigens was examined. The authors used morphometry to compare the kinetics of swine leukocyte antigen (SLA) pig thyroid xenograft rejection in control mice and mice immunized with PERV PK15 cells (porcine kidney epithelial cells), PERV SLA pig peripheral blood lymphocytes (PBL), PERV virions purified from PK15 cells, and PERV or PERV A pseudotypes produced from infected human 293 cells. The tempo of rejection for cellular xenografts of PERV A pseudotype-producing human 293 cells, uninfected human 293 cells, and PK15 cells in PERV-preimmunized and control mice was also compared., Results: Mice immunized with PK15 cells rejected pig thyroid xenografts significantly faster at day 5 than control mice and mice immunized with pig PBL. This correlated with the amount of PERV RNA and virions produced, but not with the amount of SLA class I MHC expressed by PK15 cells. Immunization of mice with PERV virions purified from porcine PK15 cells and with PERV virions or PERV A pseudotypes produced by human 293 cells also induced accelerated xenograft rejection by 5 days. Accelerated rejection induced by virus pretreatment was CD4 T-cell dependent and restricted to PERV-expressing cellular xenografts of porcine or nonporcine origin., Conclusions: PERV acts as a significant source of xenoantigens that target porcine cellular xenografts for rejection.

The rejection of pig proislet xenografts in mice is a CD4 T cell-dependent process in which macrophages play an important role. To assess the potential for activated macrophages to act as effector cells in xenograft destruction, we have examined the relationship between proislet xenograft rejection, two principal markers of macrophage activation, transcription of inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO), and their temporal relationship to intragraft cytokine gene expression. Xenograft rejection in CBA/H mice correlated with early induction of intragraft host iNOS mRNA and marked intragraft production of NO (reactive nitrogen intermediates, RNI). Intragraft mRNA expression for IFN-gamma, IL-1beta and TNF, cytokines associated with macrophage activation, was also found. These findings suggested that activated macrophages could be contributing to xenograft destruction via local NO-mediated toxicity at the graft site. To test the role of NO in this model: (1) Q-fever antigen (QFA) was administered to recipient mice in order to induce high systemic RNI levels and (2) in another experiment, pig proislets were transplanted into iNOS-/- mice. Treatment with QFA correlated with prolonged xenograft survival at 7 days post-transplant. Splenocytes from QFA-treated, but not control mice at 7 and 22 days post-transplant, exhibited inhibition of secondary xenogeneic mouse antiporcine mixed lymphocyte reaction (MLR) that was reversed by culture with the NOS inhibitor N-methylarginine (NMA). Despite continued elevated NO production, xenograft protection was temporary with complete rejection by day 22. Evidence that locally produced NO was not contributing to rejection was seen when pig proislets transplanted into iNOS-/- mice were rejected with normal kinetics; in these animals intragraft NO production was not detected (despite porcine iNOS gene expression). Failure of activated macrophages to achieve indefinite xenograft survival suggests that other factors are also required. Macrophage potential to effect either destructive or protective roles after pig proislet xenotransplantation suggests that such functions may depend on the site and magnitude of macrophage activation. Together these findings clearly demonstrate that high NO levels in the periphery are not damaging to xenogeneic islet tissue, neither host nor donor NO production is essential for islet xenograft rejection and consequently elevated plasma RNI levels do not represent a direct marker for rejection.